Ottens Andrew K, Kobeissy Firas H, Wolper Regina A, Haskins William E, Hayes Ronald L, Denslow Nancy D, Wang Kevin K W
Center for Neuroproteomics and Biomarker Research, University of Florida, Gainesville, Florida 32610, USA.
Anal Chem. 2005 Aug 1;77(15):4836-45. doi: 10.1021/ac050478r.
Differential proteomic analysis has arisen as a large-scale means to discern proteome-wide changes upon treatment, injury, or disease. Tandem protein separation methods are required for large-scale differential proteomic analysis. Here, a novel multidimensional platform for resolving and differentially analyzing complex biological samples is presented. The platform, collectively termed CAX-PAGE/RPLC-MSMS, combines biphasic ion-exchange chromatography with polyacrylamide gel electrophoresis for protein separation, quantification, and differential band targeting, followed by capillary reversed-phase liquid chromatography and data-dependent tandem mass spectrometry for quantitative and qualitative peptide analysis. CAX-PAGE provides high protein resolving power with a theoretical peak capacity of 3570, extendable to 7600, a wide protein mass range verified from 16 to 273 kDa, and reproducible differential sample comparison without the added expense of fluorescent dyes and imaging equipment. Demonstrated using a neuroproteomic model, CAX-PAGE revealed an increased number of differential proteins, 137, compared with 82 found by 2D difference gel electrophoresis. When combined with RPLC-MSMS for protein identification, an additional quantification step is performed for internal validation, confirming a 2-fold or greater change in 89% of identified differential targets.
差异蛋白质组学分析已成为一种大规模手段，用于识别治疗、损伤或疾病后蛋白质组范围内的变化。大规模差异蛋白质组学分析需要串联蛋白质分离方法。在此，我们提出了一种用于解析和差异分析复杂生物样品的新型多维平台。该平台统称为CAX-PAGE/RPLC-MSMS，它将双相离子交换色谱与聚丙烯酰胺凝胶电泳相结合，用于蛋白质分离、定量和差异条带靶向，随后进行毛细管反相液相色谱和数据依赖串联质谱分析，以进行肽段的定量和定性分析。CAX-PAGE具有高蛋白质分辨能力，理论峰容量为3570，可扩展至7600，可验证的蛋白质质量范围为16至273 kDa，并且无需额外的荧光染料和成像设备费用即可进行可重复的差异样品比较。使用神经蛋白质组学模型进行验证，CAX-PAGE显示差异蛋白质数量增加至137个，而二维差异凝胶电泳仅发现82个。当与RPLC-MSMS结合用于蛋白质鉴定时，会执行额外的定量步骤进行内部验证，确认89%的已鉴定差异靶点有2倍或更大的变化。
